Low-molecular-weight compounds (e.g., drugs) having toxicity or drug efficacy act in vivo on biomolecules such as proteins to exhibit bioactivity. Examining in vivo or intracellular distribution of target biomolecules on which low-molecular-weight compounds act, specifying the target biomolecules, analyzing specific sites at which low-molecular-weight compounds act, as well as elucidating the mechanism for the expression of bioactivity is extremely important for the development of effective therapeutic methods and remedies and life research that underlies such developments.
Regarding methods for examining the in vivo or intracellular distribution of target biomolecules, molecular imaging using radioactive compounds, phosphorescent compounds or fluorescent compounds, and Raman imaging for detecting scattered light of the biomolecule themselves are known. In vivo or intracellular molecular imaging is an important technique for understanding conditions of the disease status and pharmacokinetics and the like, and has recently been undergoing rapid development. Raman imaging involves detecting Raman scattering light from a sample irradiated by a laser and then imaging the distribution, by using the Raman spectroscopy method. Molecular imaging involves the use of radioactive compounds, phosphorescent compounds, or fluorescent compounds. On the other hand, Raman imaging involves the use of low-molecular-weight compounds that are nonradioactive and have only a slight effect on target molecules, and thus enables convenient direct examination of dynamic cell states. It has been reported that when alkyne or the like having a carbon-carbon triple bond is used as a label, imaging with higher sensitivity can be achieved with a minimal effect on target molecules (Non-patent Document 1). Non-patent Document 1 describes incorporating a nucleic acid analog, 5-ethyl-2′-deoxyuridine (EdU) into cells, and confirming the incorporation thereof to cell nuclei by using Raman microscope imaging (see Non-patent Document 1, page 6103, FIG. 2, and FIG. 4). In Non-patent Document 1, Raman images are obtained with wavenumbers, with which a Raman peak unique to label is obtained. Therefore, the thus obtained image corresponds to the spatial intensity distribution of the Raman peak with specific wavenumber.
Regarding the method of searching a low-molecular-weight compound such as a drug and a biomolecule which is the target of the compound and then identifying the binding site, LC-MS combining a liquid chromatograph with a mass spectrometer is used. A sample is fractionated by LC, and then the fractionated sample is subjected sequentially to MS and MS/MS analysis in an exhaustive manner, so as to specify the target biomolecule or identify the binding site. In MS analysis, the target biomolecule is searched for based on a mass shift resulting from the binding of the low-molecular-weight compound. Further, information such as the amino acid sequence of the peptide can be acquired by MS/MS analysis, and thus the binding site can be identified.
In order to identify a target biomolecule within cells by an analytical method such as LC-MS, the following series of steps are required: (1) incorporate a low-molecular-weight compound into cells and bind the low-molecular-weight compound to an intracellular target biomolecule; (2) disrupt the cells, (3) detect the target biomolecule in the cell disruption solution, and (4) analyze and specify the target biomolecule; or, (1) disrupt the cells, (2) mix the cell disruption solution with a low-molecular-weight compound to bind to a target biomolecule, (3) fractionate the cell disruption solution, and (4) analyze and specify the target biomolecule. Moreover, a method for specifying and/or identifying the binding site of a biomolecule and a low-molecular-weight compound requires the following steps: (1) bind a low-molecular-weight compound to a biomolecule, (2) fragment the biomolecule bound to the low-molecular-weight compound, (3) detect the bound fragment, and (4) analyze the bound fragment to identify the binding site.
However, regarding complex samples obtained via the above steps, an exhaustive search for a biomolecule using LC-MS, sequencing, and specifying the binding site requires tremendous time and also errors are likely to arise. In addition, when the binding mode of a low-molecular-weight compound and a biomolecule is unknown, it is, in principal, impossible to search for the target molecule based on a predicted mass shift. A method (CE-MS) using a capillary electrophoretic device instead of a liquid chromatograph has also been devised. However, as with LC-MS, this method requires exhaustive detection and, therefore, an extremely large number of objects must be analyzed, and prolonged and complicated analysis procedures are required.
As a technique for selectively subjecting an intracellular target molecule to analysis such as mass spectrometry, a method comprising affinity purification using a low-molecular-weight compound bound to a carrier in order to separate and purify the target molecule has been developed and is used widely. Moreover, a method for specifying a bound target biomolecule by: generating a covalent bond using a functional group reactive to the target biomolecule; and examining a radioactive, phosphorescent, or fluorescent compound or the like introduced in advance into the low-molecular-weight compound is used. Regarding a technique for specifying and/or identifying a binding site of a target molecule, a method comprising introducing a fluorophore into the low-molecular-weight compound and observing the same is used widely. For example, regarding a method for specifying and/or identifying the binding site of a labeled drug and a protein, a method using a xanthine dye as a fluorophore (rhodamine, fluorescein, or rodol), a cyanine dye, a coumarin dye, or a composite dye as a label for the drug has been reported (Patent Document 1).
When a radioactive compound is used as the low-molecular-weight compound there is no effect on the activity of the target molecule since radio isotopes basically have identical chemical properties. However, facilities in which the method can be used are limited to those in which radiation can be controlled. Further such method strictly restricts the step of identifying the binding site and is not convenient. Unlike methods using radioactive compounds, there are very few restrictions on carrying out methods that involve direct binding of a phosphorescent compound or a fluorescent compound having a large molecular weight to the target molecule. However, since the molecular weight of a fluorophore becomes relatively higher than that of the low-molecular-weight compound, such method is problematic in that the activity or binding properties of the low-molecular-weight compound can be affected. For example, whereas fluorouracil (5-FU), a type of anticancer agent, has a molecular weight of 130, Rhodamine 6G, a typical fluorophore, has a molecular weight of 479. When 5-FU is labeled with Rhodamine 6G, the bioactivity of the anticancer agent, 5-FU, can be affected by the fluorescent label. Further, flavagline, an anticancer agent extracted from an Aglaia plant, inhibits cell growth in a cancer-cell-specific manner and is not likely to cause side effects. Therefore, attempts have been made to elucidate the in vivo mode of action thereof. However, it is reported that when flavagline is labeled with a fluorophore, the drug activity decreases to 1/40 or less of its previous level. Non-patent Document 2 (page 5180, right column) describes that while the IC50 (concentration at which flavagline suppress cell growth by 50%) of flavagline is 3 nM, the IC50 of flavagline labeled with fluorescence decreases to 130 nM. Non-patent Document 3 reports that the molecule 16F16, which binds to a target protein, loses its activity when modified with a fluorophore (Non-patent Document 3, page 901, right column, lines 13-17).
A modified version of the above labeling methods has been reported, which involves binding a low-molecular-weight compound (alkyne) containing an alkynyl group as a functional group to a target biomolecule, introducing a fluorophore via a click reaction, and degrading and fragmenting the target biomolecule using an enzyme and the like (see Non-patent Document 3, page 902, FIG. 3). The use of this method leads to decreased detrimental effects such as dissipation of the activity of a target protein. However, this method is problematic in that procedures are complex, nonspecific binding reactions occur, a catalyst such as copper is needed, and there is loss of the target molecule due to reaction procedures. Therefore, when the amount of a sample is insufficient, there are limits to apply this method in practice. Regarding methods for searching for intracellular post-translational modification of a protein, examples using a click reaction include a report of incorporating a palmitoyl lipid into cells, modifying the same with a fluorophore via a click reaction, and then specifying a protein that binds to the lipid using fluorescence analysis (Non-patent Document 4) and a report of introducing a biotin tag into a farnesyl lipid via a click reaction and then detecting the same with streptavidin (Non-patent Document 5). However, these methods also have the problems above associated with click reactions.
Unlike techniques that involve searching a target molecule via a label such as a radioactive substance or a fluorophore, the Raman spectroscopy method can detect a target molecule without using any label by a based on molecular vibration information. There are no limitations on Facilities to carry out Raman spectroscopy and the method does not affect the activity or the binding properties of the low-molecular-weight compound. Thus, the combination of Raman spectroscopy and LC-MS may constitute a new detection technique that overcomes the various problems described above. To date, an example of analyzing lysozyme using a combination of a Raman spectroscopic apparatus and a matrix assisted laser desorption/ionization mass spectrometer has been reported (Patent Document 2, column 27, FIG. 31 and claim 21). However, the object to be achieved by the invention described in Patent Document 2 is to increase the sensitivity of Raman spectroscopy, and Patent Document 2 discloses a technique for aggregating a sample in an isolated state. The reason a mass spectrometer is used in Patent Document 2 is to re-confirm the results confirmed by Raman spectroscopy using a different method. Therefore, the method of Patent Document 2 is substantially different from that of the present invention for specifying a biomolecule that binds to a low-molecular-weight compound and identifying the binding site.